Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3).

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni(2+)-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC(50) of 3.6 and 1.9 microM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method.

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure-activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC(50)=1.56-3.13 microM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 microM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides.

Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris.

Beta-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant beta-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding beta-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, beta-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l(-1). The beta-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml(-1) with barley beta-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized beta-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45 degrees C, respectively.

Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli.

Antimicrobial peptides are promising candidates for therapeutic and industrial application owing to their broad spectrum. In this work, a cost-effective method for expression of a potent antimicrobial peptide, bovine lactoferricin derivative LfcinB15-W4,10, has been developed. The oligonucleotide encoding the peptide was linked to generate different oligomeric oligonucleotide segments containing from one to nine but eight tandem copies which was inserted individually to the E. coli expression vector pET32a. The thioredoxin fusion peptides were successfully expressed and detected with different molecular weight on SDS gel, respectively. Among the monomer and other multimeric peptides, the tetramer was expressed at the highest level. After purification, more than 10 mg of tetramer with 99% purity was obtained from 1 l culture and exhibited similar antimicrobial activity as synthetic LfcinB15-W4,10 monomer. The expression system in this study provides a potential production method for lactoferricin derivatives and other antimicrobial peptides in research and industrial applications.

Cloning of beta-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3).

Beta-1,3-1,4-glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone beta-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml(-1) for 1% (w/v) barley beta-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg(-1) for these two substrates. The expression level of recombinant beta-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40 degrees C, respectively.